1. Field of the Invention
The present invention relates to a polyacrylamide gel for electrophoresis, a polyacrylamide gel electrophoresis method using the gel, a method of producing the gel, and an acrylamide compound.
2. Description of the Related Art
Some kinds of biological enzymes have serine, threonine, and tyrosine residuals at a specific site such as an active center or an allosteric site and an enzyme activity is regulated by phosphorylation or dephosphorylation of their hydroxyl groups by an enzyme called as kinase. Further, there are enzymes whose activity is regulated by phosphorylation or dephosphorylation of amino or imino groups of lysine, arginine and histidine, or carboxyl groups of aspartic acid and glutamic acid.
As a metabolism system regulated by such phosphorylation-dephosphorylation, a glycogen synthesis and decomposition system is well known. This metabolism system is cascade-controlled and regulated mainly by phosphorylation-dephosphorylation.
Recently, it has been made clear that this phosphorylation-dephosphorylation plays an important role in a metabolism system relevant to disease. For example, it is considered that the abnormality of the phosphorylation-dephosphorylation is one of causes of cell canceration. That is, advancement and cease of cell cycles are controlled by phosphorylation or dephosphorylation of various enzymes (proteins), and cyclin and cyclin-dependent kinase (CDK) are relevant to the phosphorylation or dephosphorylation, but if such a mechanism is damaged, phosphorylation (dephosphorylation) is disordered and accordingly, abnormal propagation of cells is to be induced.
In addition, the following have been made clear: proteinkinase C is relevant to degranulation of mast cells to be a cause of allergy disease such as atopic dermatitis and hay fever; neurofibrillary change occurred in the brain of a patient suffering from Alzheimer disease is due to phosphorylated tau protein.
Accordingly, understanding of the phosphorylation-dephosphorylation state of protein may possibly be useful for not only search of gene expression of cells in body tissues and enzyme activity evaluation but also diagnosis and treatment of disease.
However, methods of specifying phosphorylated protein or dephosphorylated protein employed conventionally have various disadvantages. For example, an enzyme immunization method has an advantage that analysis is possible even if the amount of a protein sample as a target is slight. However, it is difficult to sufficiently obtain a needed antibody and in the case where the target protein is in several kDa or less, it is impossible to prepare an antibody to be bonded at the phosphorylation site of the protein.
A method for detecting specific bonding to protein by using phosphoric acid labeled with a radioactive isotope 32P is supposed to be possible. However, carefulness is certainly required in handling the radioactive isotope and it is also necessary to dispose and treat a waste solution.
Further, it is supposed to be possible to apply a two-dimensional electrophoresis method since phosphorylated protein and dephosphorylated protein have different electric charges separately. However, since there are no less than 5 kinds of amino acids such as aspartic acid and lysine having electric charge in the side chains among 20 kinds of amino acids composing protein, it is assumed that the electric charge alteration by the phosphorylation or dephosphorylation is small relative to the electric charge of the protein itself. In such a case, it is difficult for the electrophoresis method depending on the electric charge to detect phosphorylation or dephosphorylation. Particularly, in the case of analyzing a biological sample, since many types of proteins are contained in the sample, the detection is impossible only on the basis of slight band position change. In addition, if a radioactive isotope is employed for spot specification, the above-mentioned problem occurs.
WO03/053932 discloses a compound having specific and high coordination ability for a phosphate group, and a method of specifying phosphorylated peptide by adding the compound to a sample and then changing the charge of the phosphated peptide to carry out electrophoresis. However, there is no description of a compound having a side chain including an acrylamide structure, and a more sensitive phosphorylated peptide detection method is required.